Rat osteosarcoma cell line OSR-6

ABSTRACT

The present invention relates to a novel cell line isolated from a rat osteosarcoma wherein the cell line has the following characteristics: a) normal p53 gene as compared to a non-tumorigenic Sprague-Dawley rat cell control; b) normal RB-1 gene as compared to a non-tumorigenic Sprague-Dawley rat cell control; c) a normal c-myc gene as compared to a non-tumorigenic Sprague-Dawley rat cell control; d) a normal c-fos gene as compared to a non-tumorigenic Sprague-Dawley rat cell control; e) a deregulated immediate early gene response; f) a canalicular network MATRIGEL™ growth pattern; g) tumorigenic in congenitally athymic mice; h) low levels of alkaline phosphatase activity; i) an ability to produce one or more of the following growth factors: 1) a nonheparin binding growth factor, 2) a first heparin binding growth factor, 3) a second heparin binding growth factor, 4) a third heparin binding growth factor, 5) a fourth heparin binding growth factor, 6) a fifth heparin binding growth factor, 7) a sixth heparin binding growth factor; and j) an ability to be serially propagated greater than sixty population doublings. 
     The present invention also relates to growth factors having the characteristics of growth factors isolated from the rat osteosarcoma cell line. 
     The present invention further relates to a process for producing such growth factors comprising growing the cells of the present invention in a culture medium and recovering the growth factors.

TECHNICAL FIELD

This invention relates generally to a novel cell line, and specificallyto a novel rat osteosarcoma cell line, as well as to certain growthfactors produced by the cell line.

BACKGROUND OF THE INVENTION

Polypeptide growth factors play a key role in regulating the developmentof multicellular organisms and in the processes of tissue maintenanceand repair. (Cross and Dexter (1991) Cell, Vol. 64, pp. 271-280; andAaronson (1991) Science, Vol. 254, pp. 1146-1153.) At the cellularlevel, growth factors are involved in regulating proliferation and theprogressive acquisition of the differentiated phenotype. Growth factorsare capable of stimulating cellular proliferation as well as inhibitingcellular proliferation and many growth factors have been found to bemultifunctional (Sporn and Roberts (1988) Nature, Vol. 332, pp.217-219). The highly coordinated functions of growth factors is perhapsbest exemplified in the development of the hematopoiectic cell system(Metcalf (1989) Nature, Vol. 339, pp. 27-30) where a limited number ofstem cells give rise to a larger population of developmentallyrestricted progenitor cells. These progenitors cells are furtherstimulated to proliferate and differentiate into mature lymphoid,erythroid and myeloid cells. A balance between cell types and numbers ofcells must be maintained throughout the developmental cascade. Thisrequires the concerted actions of growth factors which commit a cell(now developmentally restricted) along a particular cell lineage, ofgrowth factors which stimulate the proliferation of committed cells, andfinally, of growth factors which promote the differentiation of thecommitted cells and inhibit the proliferation of the mature, fullydifferentiated cells.

Tumor cells represent naturally occurring examples of cells where theprocesses that control cellular proliferation and differentiation havebeen uncoupled (Cross and Dexter (1991) Cell, Vol. 64, pp. 271-280;Aaronson (1991) Science, Vol. 254, pp. 1146-1153). The observation thatmany types of tumor cells secrete growth factors suggests that thesefactors can contribute to the tumorigenic process as well as normalcellular processes. Tumor cells have been found to secrete autocrinegrowth factors which stimulate the proliferation of the tumor cellsthemselves and paracrine growth factors which stimulate surroundingcells to secret factors promoting the proliferation of the tumor cells.Paracrine factors can also stimulate the surrounding cells to provide acellular environment promoting the survival of the tumor cells. Forexample, many types of tumor cells secrete growth factors that recruitendothelial cells and stimulate their proliferation and differentiationresulting in a new vasculature supplying nutrients for the tumor cells(Liotta et al., (1991) Cell, Vol. 64, pp. 327-336).

OBJECTS OF THE PRESENT INVENTION

It is an object of the present invention to provide a novel cell line.

It is also an object of the present invention to provide novel growthfactors having the characteristics of growth factors produced by thecell line.

It is also an object of the present invention to provide a process forproducing novel growth factors from the novel cell line.

SUMMARY OF THE INVENTION

The present invention relates to a novel cell line isolated from a ratosteosarcoma wherein the cell line has the following characteristics: a)normal p53 gene as compared to a non-tumorigenic Sprague-Dawley rat cellcontrol; b) normal RB-1 gene as compared to a non-tumorigenicSprague-Dawley rat cell control; c) a normal c-myc gene as compared to anon-tumorigenic Sprague-Dawley rat cell control; d) a normal c-fos geneas compared to a non-tumorigenic Sprague-Dawley rat cell control; e) aderegulated immediate early gene response; f) a canalicular networkMATRIGEL™ growth pattern; g) tumorigenic in congenitally athymic mice;h) low levels of alkaline phosphatase activity; i) an ability to produceone or more of the following growth factors: 1) a nonheparin bindinggrowth factor, 2) a first heparin binding growth factor, 3) a secondheparin binding growth factor, 4) a third heparin binding growth factor,5) a fourth heparin binding growth factor, 6) a fifth heparin bindinggrowth factor, 7) a sixth heparin binding growth factor; and j) anability to be serially propagated greater than sixty populationdoublings.

The present invention also relates to growth factors having thecharacteristics of growth factors isolated from the rat osteosarcomacell line.

The present invention further relates to a process for producing suchgrowth factors comprising growing the cells of the present invention ina culture medium and recovering the growth factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b and 1c Heparain agarose chromatography of OSR-6 conditionedmedium. FIG. 1a at 280 nm of the pooled fractions was recorded, and NaClconcentration was determined by conductivity measurements of selectedfractions. FIG. 1b mitogenic activity of the pooled fractions usingOSR-2 cells. FIG. 1c mitogenic activity of the pooled fractions usingNIH-3T3 cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "ATCC" means American Type Culture Collection.

As used herein, "OSR-6" refers to the novel cell line of the presentinvention, ATCC Accession No. CRL 11069.

As used herein, "OSR-2" refers to ATCC Accession No. CRL 11065.

As used herein, "w/v" and "v/v" mean percent by weight and percent byvolume, respectively.

As used herein, "tumorigenic" means an ability to generate tumors in asuitable animal host.

As used herein, "heparin agarose elution property of XM NaCl" means themolarity of NaCl used to achieve elution of a protein from a heparinagarose column when a conditioned media comprising the protein issubjected to the heparin agarose column procedure described below,wherein X is a numerical value.

The present invention relates to a novel cell line isolated from a ratosteosarcoma wherein the line has the following characteristics: a)normal p53 gene as compared to a non-tumorigenic Sprague-Dawley rat cellcontrol, b) normal RB-1 gene as compared to a non-tumorigenicSprague-Dawley rat cell control; c) a normal c-myc gene as compared to anon-tumorigenic Sprague-Dawley rat cell control; d) a normal c-fos geneas compared to a non-tumorigenic Sprague-Dawley rat cell control; e) aderegulated immediate early gene response; f) a canalicular networkMATRIGEL™ growth pattern; g) tumorigenic in congenitally athymic mice;h) low levels of alkaline phosphatase activity; i) an ability to produceone or more of the following growth factors: 1) a nonheparin bindinggrowth factor, 2) a first heparin binding growth factor, 3) a secondheparin binding growth factor, 4) a third heparin binding growth factor,5) a fourth heparin binding growth factor, 6) a fifth heparin bindinggrowth factor, 7) a sixth heparin binding growth factor; and j) anability to be serially propagated greater than sixty populationdoublings.

Preferably, the non-heparin binding growth factor has an inability tobind heparin and an ability to stimulate the proliferation of fibroblastcells and osteoblast cells.

Preferably the first heparin binding growth factor has a heparin agaroseelution property of from about 0 to about 0.2M NaCl, more preferablygreater than 0 to less than or equal to 0.2M NaCl, and has an ability tostimulate osteoblast cells.

Preferably, the second heparin binding growth factor has a heparinagarose elution property of from about 0.2 to about 0.4M NaCl, morepreferably greater than 0.2 and less than or equal to 0.4M NaCl, anability to stimulate fibroblast cells, and an ability to stimulateosteoblast cells.

Preferably, the third heparin binding growth factor has a heparinagarose elution property of from about 0.5 to about 0.7M NaCl, morepreferably from about 0.5 to less than or equal to 0.7M NaCl, an abilityto stimulate fibroblast cells, and an ability to stimulate osteoblastcells.

Preferably, the fourth heparin binding growth factor has a heparinagarose elution property of from about 0.7 to about 0.9M NaCl, morepreferably from greater than 0.7 to about 0.9M NaCl, and an ability tostimulate osteoblast cells.

Preferably, the fifth heparin binding growth factor has a heparinagarose elution property of from about 1.0 to about 1.2M NaCl, anability to stimulate fibroblast cells, and an ability to stimulateosteoblast cells.

Preferably, the sixth heparin binding growth factor has a heparinagarose elution property of from about 1.5 to about 1.8M NaCl, anability to stimulate fibroblast cells, and an ability to stimulateosteoblast cells.

Preferably the cell line has the characteristics of American TypeCulture Collection Accession No. CRL 11069.

The present invention also relates to growth factors having thecharacteristics of the above-identified growth factors.

The present invention further relates to a process for producing suchgrowth factors comprising growing the cells of the present invention ina culture medium and recovering the growth factors.

An alternative way of obtaining the growth factors produced by the cellline of the present invention is by isolation of the growth factor mRNAfor use by those skilled in the art for expression of the protein ofinterest. Protein expression from mRNA covers a wide variety oftechniques including PCR methodologies using a number of organisms forthe final expression of the protein including bacteria, fungus, animalcells, insect cells and plant cells as well as noncellular proteinexpression methodologies (for an overall review of molecular biologytechniques used in protein expression cloning see Sambrook et al. (1989)Molecular Cloning--A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Briefly, this procedureinvolves isolating mRNA from an osteosarcoma cell line which codes forthe growth factor, making cDNA of the mRNA, cloning the cDNA into anexpression vector, expressing the cDNA in a host, and recovering arecombinant form of the growth factor. More preferably, this procedureinvolves isolation of mRNA from the osteosarcoma cell, making cDNA fromthe mRNA, cloning of the cDNA into an appropriate expression source,expression of the protein of interest from the cDNA inserted into theexpression vector, screening the expression source for the protein ofinterest, purifying the expression clone containing the cDNA coding forthe protein of interest, and expression of this purified cDNA in anexpression vector in an appropriate expression source for large scalesynthesis of the protein of interest. Multiple technical variations ofthis general expression schemes are well understood by those skilled inthe art and all would apply.

The cell line of the present invention is useful as a biological sourcefor the growth factors of the present invention and/or the mRNA codingfor such growth factors. The growth factors of the present invention areuseful for one or more of the following: 1) treating diseases affectingthe bone and cartilage (e.g., those growth factors demonstrating anability to stimulate osteoblast cells), and/or 2) wound healing (e.g.,those growth factors demonstrating an ability to stimulate fibroblastcells).

STATEMENT OF DEPOSIT

OSR-6 has been deposited at the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, U.S.A., on Jun. 5, 1992. Thedeposited strain has been assigned Accession No. CRL 11069.

The subject cultures have been deposited under conditions that assureaccess to the cultures will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR §1.14 and 35 USC §122.The deposits are available as required by foreign patent laws incountries where counterparts of the subject application, or its progeny,are filed. However, it should be understood that Applicants' granting ofpermission to the depository to distribute samples of the deposit doesnot constitute an express or implied license to practice the inventionclaimed in any patent issuing on the subject application or any otherpatent.

The subject culture deposits will be stored and made available to thepublic in accord with the provisions of the Budapest Treaty for theDeposit of Microorganisms, i.e., they will be stored with all the carenecessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposits, and in any case, for a period of at least thirty(30) years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposits. All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

ESTABLISHMENT OF THE OSR-6 CELL LINE

A cell line, OSR-6, was established from a tumor excised from aSprague-Dawley rat with osteosarcoma. The site of the excised tissue wasthe right tibia. The tumor tissue was aseptically remove from theeuthanized animal and carefully trimmed of connective tissues. The tumorwas minced in a culture dish (100 mm diameter) containing 15 ml ofgrowth medium (10% fetal bovine serum, 90% RPMI 1640 medium (GIBCO))further supplemented with penicillin (100 units/ml, GIBCO), Fungizone(0.25 microgram/ml, GIBCO) and streptomycin (100 micrograms/ml,GIBCO),and incubated at 370° C. in an atmosphere of 5% CO₂ and 95% air with arelative humidity of approximately 95%. The adherent cells whichmigrated from the minced tumor pieces onto the surface of the culturedish were further expanded as a cell population using standard tissueculture techniques. Once the OSR-6 cell line reached confluence in atissue culture flask (150 cm² surface area), the cell line wasdesignated as having a mean population doubling of 1. At this stage, thecells were subcultured in growth medium (10% fetal bovine serum, 90%RPMI 1640 medium) with no antibiotic or antimycotic supplements. TheOSR-6 cell line was routinely tested for mycoplasma contamination byDAPI assay (Stanbridge (1981) Isr. J. Med. Sci., Vol.17, pp. 563-568)and was found to be negative.

The OSR-6 cell line was found to be tumorigenic when assayed for tumorformation in congenitally athymic (nu/nu) mice (Harlan Sprague Dawley,Inc.). OSR-6 cells at a mean population doubling 13, were injectedsubcutaneously in the mid-flank region of the animals at an innoculum ofapproximately 1×10⁷ cells per site. A total of eight sites (two peranimal) were injected. Tumors arose at 100% of the sites injected withthe OSR-6 cells.

CHARACTERISTIC OF THE OSR-6 CELL LINE

The OSR-6 cell line can be serially propagated in culture with no knownlimited life span. The OSR-6 cell line displays a limited, immatureosteoblast-like phenotype in vitro. The cells show faint staining forthe presence of alkaline phosphatase (Rodan and Rodan (1983) Bone andMineral Research, Annual 2 (Peck, ed.) pp.244-285) as detected by acytochemical assay (Sigma Chemical Company, Procedure 85). It has beenreported (Vukicevic et al., (1990) Cell, Vol. 63, pp. 437-445) thatosteoblast cells are capable of forming cell clusters exhibitingnetworks of canalicular cell processes when cultured on reconstitutedbasement membrane extracts such as MATRIGEL™ (a solublized tissuebasement membrane available from Collaborative Biomedical Products,Bedford, Mass., containing laminin, collagen type IV, heparin sulfate,proteoglycan and entactin). Fibroblasts, chondrocytes and embryonic stemcells did not demonstrate the canalicular cell processes. OSR-6 cellswhen cultured on MATRIGEL™ at a density of approximately 50,000 cellsper well of a standard 24-well tissue culture plate formed many clustersof cells with a network of canalicular cell processes. This growthpattern was very similar to the cell clusters with the characteristiccanalicular processes that were formed by the osteoblastic MC3T3-E1cells plated at an equivalent density (Sudo et al., (1983) J. CellBiol., Vol. 96, pp. 191-198; Vukicevic et al., (1990) Cell, Vol. 63, pp.437-445).

MOLECULAR CHARACTERIZATION OF THE OSR-6 CELL LINE

In order to characterize the OSR-6 cell line in terms of uniquemutations which can identify this cell line we have screened the cellline for mutations in genes which are commonly mutated in osteosarcomas.This molecular fingerprint of the OSR-6 cell provides a convenient anduseful way to identify this cell line because these mutations arecritical for the growth of these cells and is not expected to change.

In this respect human osteosarcomas have been extensively studied withrespect to mutations in the retinoblastoma (RB-1) and p53 tumorsuppressor genes (Iavarone et al., (1992) Proc. Natl. Acad. Sci. USA,Vol. 89, pp. 4207-4209; Diller et al., (1990) Mol. Cell. Biol., Vol. 10,pp. 5772-5781; Masuda et al., (1987) Proc. Natl. Acad. Sci. USA, Vol.84, pp. 7716-7719; Levine and Momand (1990) Biochem. et Byophys. Acta,Vol. 1032, pp. 119-136; Miller et al., (1990) Cancer Res., Vol. 50, pp.7950-7954; Friend et al., (1987) Proc. Natl. Acad. Sci. USA, Vol. 84,pp. 9059-9063; Mulligan et al., (1990) Proc. Natl. Acad. Sci. USA Vol.87, pp. 5863-5867; Hansen et al., (1985) Proc. Natl. Acad. Sci. USA,Vol. 82, pp. 6216-6220; Horowitz et al., (1989) Science, Vol. 243, pp.937-940; Toguchida et al., (1989) Nature, Vol. 338, pp. 156-158). RB-1mutations include point mutations, deletions, and splicing mutations(Friend et al., (1987) Proc. Natl. Acad. Sci. USA, Vol. 84, pp.9059-9063; Levine and Momand, (1990) Biochem. et Bioshys. Acta, Vol.1032, pp. 119-136; Mori et al., (1990) Oncogene Vol. 5, pp.1713-1717;Horowitz et al., (1989) Science, Vol. 243, pp. 937-940). All of thesemutations result in the production of a non-functional RB-1 gene. RB-1is believed to function by acting as a "brake" on cellular proliferationat the appropriate time in the cell cycle. Control of the RB-1 functionis achieved by selective and timely phosphorylation of RB-1 protein;phosphorylated RB-1 allows cellular proliferation while unphosphorylatedRB-1 protein inhibits cellular proliferation (Laiho et al., (1990) Cell,Vol. 62, pp. 175-185; DeCaprio et al., (1989) Cell, Vol. 58, pp.1085-1095; Buchkovich et al., (1989) Cell, Vol. 58, pp. 1097-1105; Chenet al., (1989) Cell, Vol. 58, pp.1193-1198; Furukawa et al., (1990)Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 2770-2774). RB-1 control ofcellular proliferation is one part of a closely regulated network ofcell cycle controls, which include the cell cycle control proteinsc-myc, c-fos and TGF-B (Laiho et al., (1990) Cell, Vol. 62, pp. 175-185;Moses et al., (1990) Cell, Vol. 63, pp. 245-247; Robbins et al., (1990)Nature, Vol. 346, pp. 668-671).

The p53 tumor suppressor gene was originally identified as an SV40 largeT antigen binding protein whose expression was greatly increased(approximately 5-100 fold at the protein level) during SV40 cellulartransformation (Levine and Momand, (1990) Biochem. et Biophys. Acta,Vol. 1032, pp. 119-136; Levine et al., (1991) Nature, Vol. 351, pp.453-456). The p53 gene has since been found to be mutated in a varietyof tumor types (Hollstein et al., (1991) Science, Vol. 253, pp. 49-53)including human osteosarcoma cells (Mulligan et al., (1990) Proc. Natl.Acad. Sci. USA, Vol. 87, pp. 5863-5867; Miller et al., (1990) CancerRes., Vol. 50, pp. 7950-7954; Masuda et al., (1987) Proc. Natl. Acad.Sci. USA 84, 7716-7719; Diller et al., (1990) Mol. Cell. Biol., Vol. 10,pp. 5772-5781). Mutation of the p53 gene either results in an alteredprotein which does not function normally or a complete loss of protein.Both of these mechanisms result in the absence of a functional p53protein (Halevy et al., (1990) Science, Vol. 250, pp. 113-116; Chen etal., (1990) Science, Vol. 250, pp. 1576-1580; Milner and Medcalf, (1991)Cell, Vol. 65, pp. 765-774) and cell-ular transformation. The p53protein is believed to function in several ways. The p53 protein is atranscriptional activator since p53 protein binds to specific DNAsequences (Raycroft et al., (1990) Science, Vol. 249, pp. 1049-1051;Kern et al., (1991) Science, Vol. 252, pp. 1708-1711) and contains anNH₂ -terminal acidic domain which efficiently activates thetranscription of genes in both yeast and mammalian cells (Fields andJang, (1990) Science, Vol. 249, pp. 1046-1049). The protein producedfrom mutated p53 genes does not transcriptionally activate responsivegenes (Raycroft et al., (1990) Science, Vol. 249, pp. 1049-1051). Inaddition, p53 regulates DNA replication since wild-type protein but notprotein from mutated p53 genes associates with replication complexes(Levine et al., (1991) Nature, Vol. 351, pp. 453-456; Levine and Momand,(1990) Biochem. et Biophy. Acta, Vol. 1032, pp. 119-136) and isphosphorylated by p34(cdc2)-p06 and p34(cdc2)-cyclin B complexes(Bischoff et al., (1990) Proc. Natl. Acad. Sci. USA, Vol. 87, pp.4766-4770; Milner et al., (1990) EMBO J, Vol. 9, pp. 2885-2889).

Two additional transforming genes which have been implicated in humanand non-human osteosarcoma formation are the fos and myc oncogenes. Theoncogenic variant of c-fos, v-fos, was first isolated as thetransforming gene in retroviruses which resulted in murine osteosarcomaformation (Varmus (1984) Ann. Rev. Genet., Vol. 18, pp. 553-612).Interestingly, transgenic mice carrying the v-fos oncogene display bothcartilage and osteosarcoma tumors (Wang et al., (1991) EMBO J, Vol. 10,pp. 2437-2450). Oncogenic mutation of c-fos results when the fos gene iseither over expressed or inappropriately expressed as a result of eitherviral transduction or mutation. C-fos functions in the cell, inconjunction with c-jun as the complex which binds the AP-1transcriptional activation site (Abate et al., (1990) Science, Vol. 249,pp. 1157-1161; Sassone-Corsi et al., (1988) Cold Spring Harbor Symposiaon Quantative Biology LIII, pp. 749-760).

C-myc has been found to be mutated both in radiation induced murineosteosarcomas (Sturm et al., (1990) Cancer Res., Vol. 50, pp. 4146-4153)and in primary human osteosarcoma tumors (Bogenmann et al., (1987)Cancer Res., Vol. 47, pp. 3808-3814). The myc oncogene was originallyidentified as the transforming gene in several retroviruses (v-myc) andc-myc mutations in a variety of tumor types were later identified(DePinho et al., (1987) J. Cell Biochem., Vol. 33, pp. 257-266; Varmus(1984) Ann. Rev. Genet, Vol. 18, pp. 553-612). C-myc is most commonlyamplified or translocated, both of which, along with retroviraltransduction, result in the inappropriate expression of the myc gene(Varmus (1984) Ann. Rev. Genet., Vol. 18, pp. 553-612; DePinho et al.,(1987) J. Cell Biochem., Vol. 33, pp. 257-266). Myc protein and the Maxprotein form a complex which binds the regulatory region of genes, via aunique sequence, to control their expression (Blackwood and Eisenman,(1991) Science, Vol. 251, pp. 1211-1217; Cole (1991), Cell, Vol. 65, pp.715-716). C-myc, along with c-fos are immediate early genes and arebelieved to play a central role in mitogenic signalling in the cell(Rozengurt (1986) Science, Vol. 234, pp. 161-166).

An additional way to assay c-fos and c-myc functional activity is todetermine if theses genes are transcriptionally activated followingmitogenic stimulation. C-fos and c-myc are both members of a class ofgenes called the immediate early genes (Sassone-Corsi et al., (1988)Cold Spring Harbor Symposia on Quantitative Biology LIII, pp. 749-760;Depinho et al., (1987) J. Cell. Biochem., Vol. 33, pp. 257-266). Genesin this class are transcriptionally activated following mitogenicstimuli usually within 1 hour and without the need of protein synthesis(Almendral et al., (1988) Mol. Cell. Biol., Vol. 8, pp. 2140-2148;Greenberg et al., (1986) Mol. Cell. Biol., Vol. 6, pp. 1050-1057). Sincethis is a complex pathway, alterations in immediate early genetranscription is indicative of either a mutation in the cellular pathwaywhich leads from the receptor/plasma membrane to the gene/nucleus of thecell or a mutation which leads to the autocrine expression of a growthfactor. Either of the above mutated phenotypes lead to a down regulationand subsequent inhibition of expression of the c-myc and c-fos genesfollowing serum starvation and mitogen stimulation.

CHARACTERIZATION OF TUMOR SUPPRESSOR GENES AND ONCOGENES

High molecular weight DNA was isolated from the OSR-6 cell line asfollows. Approximately 1×10⁸ cells were cultured as described aboveuntil confluent. These cells were then scraped off the tissue cultureflask into culture media, collected by centrifugation at 3300 g for 5minutes, the culture media was removed from the cell pellet and thepellet was resuspended in 9.5 ml of NET buffer (100 mM sodium chloride,10 mM Tris - pH 8.0, 1 mM EDTA). To the resuspended DNA was added 50 ulof 10 mg/ml of proteinase K (Boehringer Mannheim) and 0.5 ml of 10%(w/v) sodium dodecyl sulphate/water. This mixture was mixed well andincubated at 55° C. for one hour followed by extraction twice with anequal volume of a 50:50 mixture of phenol:chloroform. The aqueous phasewas collected by centrifugation at 3300 g for 5 minutes and re-extractedwith an equal volume of chloroform. The aqueous phase was collected bycentrifugation at 3300 g for 5 minutes, removed to a new tube and to itwas added 1 ml of 3M sodium acetate pH 5.2 and 20 ml of 100% ethanol.This solution was mixed well and incubated at -20° C. for 2 hoursfollowed by the collection of high molecular weight DNA bycentrifugation at 3300 g for 30 minutes. The high molecular weight DNAwas washed once with 5 ml of 70% ethanol/water (v/v), dried andresuspended in TE (10 mM Tris-pH 8.0 and 1 mM EDTA) at a concentrationof 1 mg/ml.

For restriction enzyme analysis, 20 ug of the above isolated highmolecular weight DNA in 20 ul TE was added to 2.5 ul of 10× restrictionenzyme buffer (1× restriction enzyme buffer consist of 50 mM Tris - pH8.0, 10 mM magnesium chloride, and 100 mM sodium chloride) and 2 ul ofrestriction enzyme (either Eco RI or Hind III at 10 units/ml both fromNew England Biolabs) and this mixture was incubated at 37° C. for 16hours. Following incubation, the above solution was added to 3 ul of 10×gel loading buffer (10× gel loading buffer is 0.25% bromophenol blue,0.25% xylene cyanol, 25% Ficoll type 400, 10 mM EDTA in water) and therestriction endonuclease generated fragments were separated by agarosegel electrophoresis. Agarose gel electrophoresis was performed asfollows. A 0.6% (w/v) agarose (Bethesda Research Laboratories) gel in 1×TBE (1× TBE consist of 0.089M Tris, 0.089M boric acid, and 0.002M EDTA)was poured in a BioRad horizontal gel electrophoresis apparatus, theabove DNA solution was loaded into a well and electrophoresis wasperformed in a 1× TBE solution for 16 hours at 20 volts. Followingelectrophoresis, the gel was stained for 1 hour in 0.5 ug/ml ethidiumbromide (Sigma Chemical Company)/water solution followed by photographyunder 302 nm ultraviolet light irradiation. The gel was then preparedfor Southern blotting as follows. The gel was soaked for 1 hour in asolution of 1.5M sodium chloride and 0.5M sodium hydroxide with constantshaking followed by an incubation for 1 hour in a solution of 1.5Msodium chloride and 1M Tris - pH 8.0 with constant shaking. The gel wasthen transferred to an LKB VacuBlot apparatus (LKB Scientific) prefittedwith a sheet of BAS-NC nitrocellulose (Schleiser and Schuel) and thetransfer of DNA from the agarose gel to the nitrocellulose membrane wasperformed under 40 cm.H₂ O of pressure using 10× SSC (10× SSC is 1.5Msodium chloride and 0.15M sodium citrate, pH 7.0) as the transfermedium. The OSR-2 DNA Southern blot was then used in a hybridizationanalysis as follows. The nitrocellulose filter was first wet in 6× SSCfollowed by prehybridization in hybridization buffer [50% formamide(molecular biology grade, Bethesda Research Laboratories), 5× Denhardt'ssolution (Denhardt's solution is 0.1% Ficoll, 0.1% polyvinylpyrrolidone,and 0.1% bovine serum albumin--pentax fraction V--all from SigmaChemical Company), 5× SSPE (20× SSPE is 3M sodium chloride, 0.2M sodiumphosphate, and 0.02M EDTA--pH 7.4), 0.1% sodium dodecyl sulphate, and100 ug/ml of denatured salmon sperm DNA (Sigma Chemical Company)] for 4hours at 42° C. with constant agitation. Molecular probes for the tumorsuppressor genes RB-1 (ATCC #57450) and p53 (Levine and Momand (1990)Biochemic et Biophysic Acta, Vol. 1032, pp. 119-136) and the oncogenesc-myc (ATCC #41008) and c-fos (ATCC #41040) were radiolabelled using anAmersham nick translation kit and [32P]dCTP (ICN) to a specific activityof 1×10(8) cpm/ug DNA by following the manufacturer's recommendations.These radiolabelled probes were then added to the hybridizationsolution/nitrocellulose filter of the prehybridization step andincubated at 42° C. for 40 hours with constant agitation. Followinghybridization, the nitrocellulose filters (blots) were first incubatedin 2× SSC and 0.1% SDS at room temperature for 1 hour followed by anincubation in 0.2× SSC and 0.1% SDS at 65° C. for 1 hour. The results ofthe hybridization experiment were visualized by autoradiography at -70°C. Following autoradiography, the films were developed and used in dataanalysis.

The molecular analysis of the OSR-6 cell line RB-1, p53, c-myc and c-fosgenes indicated that all these genes appeared normal (non-mutated,wild-type) as compared to a non-tumorigenic Sprague-Dawley rat cellcontrol at the above described level of analysis (restriction enzymedigestion and Southern blotting).

RB-1 AND p53 PROTEIN ANALYSIS

Immunological identification of the p53 and RB-1 genes were performed asfollows. 1×10⁶ OSR-6 cells were labelled for 4 hours with 100 uci/ml of[35S]methionine (TranSlabel - ICN) in methionine-free RPMI-1640 (GIBCO)media containing 10% fetal bovine serum for 4 hours at 37° C. Followinglabelling, the cells were scraped off the tissue culture plastic intothe labelling media, the cells were collected by centrifugation at 3300g for 5 minutes, followed by removal of the labelling media and the snapfreezing of the cell pellet in liquid nitrogen. The frozen cell pelletswere dissolved in 1 ml of ice-cold lysis buffer (50 mM Tris--pH 8.0, 5mM EDTA, 150 mM sodium chloride, 0.5% Nonidet P-40, and 1 mMphenylmethylsulfonylfluoride) by vigorous vortexing and incubated on icewith intermediate vortexing for 30 minutes. The lysates were clarifiedof nonsoluable material by centrifugation at 10,000g for 30 minutes, thesupernate was removed to a new tube to which was added 10 ul ofantibodies specific for either mutant p53 (Oncogene Science p53 Ab-3),normal and mutant p53 (Oncogene Science p53 Ab-1), or RB-1 (a 50:50mixture of Oncogene Science RB Ab-2 and Ab-3), and 50 ul of a ProteinA/G agarose (Boehringer Mannheim):lysis buffer (50:50). The abovemixture was incubated overnight at 4° C. with constant shaking. The celllysate was aspirated off and the antigen/antibody/protein A-G pellet waswashed one time in 1 ml of lysis buffer, one time in 1 ml of SNTE buffer(50 mM Tris-pH 7.4, 5 mM EDTA, 5% sucrose--w/v, 1% Nonidet P-40, and0.5M sodium chloride), and one time in 1 ml of RIPA buffer (50 mMTris--pH 7.4, 150 mM sodium chloride, 1% Triton X-100, 0.1% sodiumdodecyl sulphate and 1% sodium deoxycholate). Following washing, theantigen/antibody/protein A-G agarose pellet was redissolved in 25 ul ofsample buffer (62.5 mM Tris--pH 6.8, 2% sodium dodecyl sulphate, 10%glycerol , 5% 2-mercaptoethanol), boiled at 100° C. for 2-3 minutes, andapplied to a well of a 10% SDS-PAGE gel. SDS-PAGE was performed asdescribed by Laemmli (Laemmli, U.K. (1970) Nature, Vol. 227, pp.680-685) for 6-8 hours at 30 mA constant current per gel. Followingelectrophoresis, the SDS-PAGE gels were incubated in 30% methanol and10% acetic acid for at least 1 hour, impregnated with ENHANCE(NEN-DuPont) according to the manufacturers recommendations, dried ontoWhatman 3 mm paper using a BioRad gel dryer set at 60° C. for 2 hoursand autoradiography at -70° C. was performed.

The results of the p53 and RB-1 gene analysis of the OSR-6 cell lineindicated that both p53 and RB-1 genes were normal both in their levelof expression and the gene's physical characteristics.

IMMEDIATE EARLY GENE EXPRESSION FOLLOWING MITOGEN STIMULATION

In order to characterize the immediate early gene (c-myc and c-fos)transcription following mitogen stimulation in the OSR-6 cell line thefollowing experiments were performed. Approximately 1×10⁸ cells weregrown to 70% confluence in a tissue culture flask in 25 ml of standardgrowth media. The cells were washed 2 time with serum-free mediafollowed by the addition of 25 ml of serum-free culture media andincubated for 12-16 hours at 37° C. under an atmosphere of 10% carbondioxide in an incubator. To the serum-starved cells was added 3 ml (10%)of fetal bovine serum and 10 ug/ml of cycloheximide and the cells wereincubated as above for 3 hours. One group of cells was not mitogenstimulated and remained serum-starved. RNA from the mitogen stimulatedand non-stimulated cells was isolated using the RNAzol (Cinna/BiotecxInc.) methodology according to the manufacturer's recommendation.Briefly, 1×10⁸ cells were lysed in situ with 10 ml of RNAzol, the lysatewas collected, 1 ml of chloroform was added to the lysate, the sampleswere vortexed vigorously for 15 seconds, and the mixture was thencentrifuged at 12,000g (4° C.) for 15 minutes. The upper (aqueous) phasewas transferred to a new tube, an equal volume of isopropanol was addedto it, the samples were cooled to -20° C. for 45 minutes, followed bypelleting of the RNA by centrifugation at 12,000g (4° C.) for 15minutes. The pelleted RNA was washed once with ice-cold 70%ethanol/water, dried, and resuspended in RNAse-free water at 20 ug/4.5ul. RNA formaldehyde agarose gel electrophoresis was performed asdescribed (Sambrook et al., (1989) Molecular Cloning, Cold Spring HarborPress, Cold Spring Harbor, N.Y.). Briefly, 20 ug of total cellular RNAwas denatured by heating to 55° C. for 15 minutes in denaturation buffer[4.5 ul RNA solution, 2.0 ul 10× RNA gel buffer (0.2M MOPS--pH 7.0, 50mM sodium acetate, and 10 mM EDTA), 3.5 ul formaldehyde and 10.0 ulformamide] followed by the addition of 2 ul of loading buffer (50%glycerol, 1 mM EDTA, 0.4% bromophenol blue, and 0.4% xylene cyanol) andloading of the sample into a well of the formaldehyde gel (1% agarose,20 mM MOPS--pH 7.0, 5 mM sodium acetate, 1 mM EDTA and 2.2Mformaldehyde). Electrophoresis was performed at 30 volts (constantvoltage) for 16 hours. Following electrophoresis the gel was stainedwith ethidium bromide (0.5 ug/ml in water) for 1 hour, destained inwater for 1 hour, and photographed under 300 nm ultraviolet light usinga Foto/Prep I (Fotodyne) transilluminator. Following photography, thegel was transferred to nitrocellulose (Schleicher & Schuell, BA-S NC)using a LKB Vacugene vacublotting apparatus operating at 50 cm.H₂ O witha 20× SSC (3M sodium chloride and 0.3M sodium citrate--pH 7.0) fluidtransfer medium. Following transfer, the RNA was fixed to thenitrocellulose filter by UV irradiation using a Stratalinker (StratageneInc.) UV crosslinker at 0.12 Joules/cm². Following RNA fixation, theNorthern blots were used in probe hydridization studies followingpreviously described procedures (Sambrook et al., (1989) MolecularCloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) that weremodified as follows. The probes of interest, c-fos (ATCC #41040) orc-myc (ATCC #41008) were radioactively labelled by using an Amershamnick translation kit following the manufacturer's recommendations.Briefly, 1 ug of probe DNA was incubated with 1× nick translationbuffer, 50 uCi alpha [³² P]-dCTP (NEN), and polymerase mix in a totalvolume of 20 ul at 15° C. for 2 hours followed by the addition of 80 ulof 1× STE (100 mM sodium chloride, 10 mM Tris--pH 8.0, and 1 mM EDTA).Separation of the incorporated from non-incorporated nucleotides wasachieved using a Biospin column (BioRad). Typically 1 ug of probe had aspecific activity of greater than 1 ×10⁸ dpm. Following nicktranslation, the probe was boiled for 10 minutes and added to aprehybridized filter (4 hours in hybridization solution at 42° C.) in 10ml of hybridization solution [6× SSPE (3M sodium chloride, 0.2M sodiumphosphate--pH 7.4 and 20 mM EDTA), 5× Denhardt's solution (1% Ficoll, 1%polyvinyl-pyrrolidone, and 1% BSA--Pentax fraction V), 0.5% sodiumdodecyl sulfate, 100 ug/ml denatured--sonicated salmon sperm DNA, and50% formamide] and incubated for 48 hours at 42° C. Followinghybridization the filters were washed at room temperature in 2×SSC--0.1% SDS followed by a wash at 65° C. with 0.2× SSC--0.1% SDS. Thefilters were then dried, mounted to 3 MM paper (Whatman) andautoradiography at -80° C. was performed using intensifying screens(NEN).

The OSR-6 cell line displayed an altered immediate early gene responsewith a constitutive expression of c-myc, even in the absence of addedgrowth factor, and an absence of transcriptional upregulation of thec-fos gene in response to mitogen stimulation.

TABLE 1 Characterizations of the OSR-6 cell line

The OSR-6 cell line has the following characteristics;

a. a normal p53 protein/gene as compared to a non-tumorigenicSprague-Dawley rat cell control

b. a normal RB-1 gene/protein as compared to a non-tumorigenicSprague-Dawley rat cell control

c. a normal c-myc gene as compared to a non-tumorigenic Sprague-Dawleyrat cell control

d. a normal c-fos gene as compared to a non-tumorigenic Sprague-Dawleyrat cell control

e. a deregulated immediate early gene response

f. a canalicular network MATRIGEL™ growth pattern

g. tumorigenic in congenitally athymic mice

h. low levels of alkaline phosphatase activity

i. production of a non-heparin binding growth factor

j. production of a first heparin binding growth factor

k. production of a second heparin binding growth factor

l. production of a third heparin binding growth factor

m. production of a fourth heparin binding growth factor

n. production of a fifth heparin binding growth factor

o. production of a sixth heparin binding growth factor

p. an ability to be serially propagated greater than sixty populationdoublings.

USE OF THE OSR-6 CELL LINER AS A SOURCE OF GROWTH FACTORS

The ability of a growth factor to stimulate, or inhibit DNA synthesis ina serum-starved quiescent cell is perhaps the most widely studiedresponse to growth factors. This ability to stimulate, or inhibit themitogenic response of a target cell is an indicator of the growthfactor's ability to stimulate, or inhibit cellular proliferation. Themitogenic response of cells can be quantitated using a [³ H]thymidineincorporation assay as previously described (Isfort (1990) Somatic CellMolec. Genet., Vol. 16, pp. 109-121). Briefly, target cells are platedat 2×10³ cells/well in a 96 well microtiter plate and incubatedovernight in growth medium to allow the cells to attach. The growthmedium is removed and the cells are washed three times with phosphatebuffered saline (GIBCO) followed by an 8 hour incubation in 50 ul/wellof serum-free medium. Mitogens such as serum and various growth factorsare added to the serum starved cells in a volume of 50 ul of medium,with serum-free medium serving as a control. After 16 hours incubation,the cells are exposed to 1 μCi [³ H]thymidine for 6 additional hours ofincubation. The cells are then harvested and lysed on glass fiberfilters using a PHD cell harvester (Cambridge Technology, Inc.) and theradioactivity in the samples was assayed by scintillationspectrophotometery.

To test for the secretion of mitogenic growth factors by OSR-6 cells,serum-free conditioned medium was harvested from cell cultures. OSR-6cells were grown to confluency in a 150 cm² tissue culture flask. Thegrowth medium was removed and the cell monolayer was rinsed three timeswith approximately 15 ml of phosphate buffered saline. The cells werethen incubated in 25 ml of serum free medium for approximately 24 hours,and the conditioned medium was harvested and clarified by centrifugationfor 15 minutes at 2000 xg to remove cells and cellular debris. To gainsome information on the types of growth factors produced by OSR-6 cells,the clarified conditioned medium was fractionated by heparin agarosechromatography (type I heparin agarose, purchased from Sigma ChemicalCompany). The binding properties of a variety of growth factors toheparin has been reported (Shing et al., (1984) Science, Vol. 223, pp.1296-1298; Klagsbrun and Shing (1985) Proc. Natl. Acad. Sci. USA, Vol.82, pp. 805-809; Hauschka et al., (1986) J. Biol. Chem., Vol. 261, pp.12665-12674), thus allowing an empirical classification of the types ofgrowth factors. After collecting the flow through fraction of theconditioned medium, heparin binding factors were eluted in a stepwiseapplication of 0.2M NaCl, 1.0M NaCl, and 2.0M NaCl in 25 mM Tris buffer,pH 8.0. All fractions were dialyzed versus water and lyophilized. Foranalysis of mitogenic activity, the lyophilized fractions wereresuspended in 2.0 ml of serum free medium and sterilized by centrifugalfiltration (Centrex filters, Schleicher and Schuell).

A panel of target cells was used which incorporated osteoblastic,fibroblastic and multipotential mesenchymal cell types. These cell linesinclude the osteoblastic OSR-2 cells (ATCC Accession No. CRL11065); theosteoblastic MC3T3-E1 cells (Sudo et al., (1983) J. Cell Biol.96:191-198); the fibroblastic MRC-5 (American Type Culture Collection,CCL 171); the fibroblastic NIH-3T3 (American Type Culture Collection,CRL 1658); the multipotential cell line, C3H10T1/2, clone 8 (AmericanType Culture Collection, CCL 226); and the multipotential cellpopulation isolated from neonatal rat muscle (designated NRM) accordingto slight modifications of the procedure describe by Sampath et al.,(1984) Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 3419-3423. Briefly, thetricep muscles were aseptically isolated from euthanized newbornSprague-Dawley rats and cleaned of connective and vascular tissue. Themuscles were minced and cultured in 15 ml of 10% fetal bovine serum, 90%CMRL-1066 medium (GIBCO) supplemented with antibiotics and antimycoticmixture (penicillin, 100 units/ml; streptomycin, 100 ug/ml; Fungizone,0.25 ug/ml, GIBCO). Once the explant cultures reached confluence in a150 cm² tissue culture flask, the NRM cell line was designated as beingat mean population doubling of 1. The NRM cell line is capable offorming multinucleate myotubes in confluent cultures and can bestimulated by TGF-B1 to differentiate into chondrocyte-like cells(Seyedin et al., (1985) Proc. Natl. Acad. Sci. USA, Vol. 82, pp.2267-2271). The mitogenic responses elicited by the heparin agarosefractionated conditioned medium from OSR-6 cells were compared to anumber of known growth factors. All growth factors were purchased fromGIBCO BRL, Life Technologies, Inc., and were tested over a 3-4 logconcentration range incorporating the effective concentrations suggestedby the supplier. The growth factors were human recombinant plateletderived growth factor-AB heterodimer (PDGF-AB); human recombinantplatelet derived growth factor-AA homodimer (PDGF-AA); human recombinantplatelet derived growth factor-BB homodimer (PDGF-BB); human recombinantacidic fibroblast growth factor (aFGF); human recombinant basicfibroblast growth factor (bFGF); human recombinant epidermal growthfactor (EGF); human recombinant insulin-like growth factor I (IGF-I);human recombinant insulin-like growth factor II (IGF-II); humanrecombinant transforming growth factor beta, type 1 (TGF-B1); humanrecombinant interleukin 1-beta (IL-1B); recombinant murine leukemiainhibitory factor (LIF); recombinant murine tumor necrosis factor alpha(TNF-a). In addition, a mixture of bone morphogenic proteins (BMP-2,BMP-3, BMP-4, and BMP-7) as isolated from bovine bone by Koenig et al.,(1991) J. Bone Mineral Res., Vol. 6, p. S206 was included in theanalysis. The BMPs have been shown to induce the formation of cartilageand bone in vivo (reviewed by Wozney (1989) Progress in Growth FactorResearch, Vol. 1, pp. 267-280) and heparin affinity chromatography wasused in the purification of the bone derived BMP mixture. The results ofthese mitogenicity assays are summarized in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Mitogenesis Results                                                           Target Cell Line                                                                      OSR-2                                                                         (ATCC No.                                                                     CRL 11065)                                                                           MC3T3-E1                                                                            NRM C3H10TV2                                                                            MCR-5                                                                             NIH-3T3                                            (osteo-                                                                              (osteo-                                                                             (multi-                                                                           (multi-                                                                             (fibro-                                                                           (fibro-                                    Stimulus                                                                              blast) blast)                                                                              potent)                                                                           potent)                                                                             blast)                                                                            blast                                      __________________________________________________________________________    0% SERUM                                                                              0      0     0   0     0   0                                          10% SERUM                                                                             +      +     +   +     +   +                                          PDGF-AA +      +     +   +     +   ND                                         PDGF-BB +      +     +   +     +   ND                                         PDGF-AB +      +     +   +     +   +                                          EGF     +      +     +   +     +   +                                          aFGF    +      +     +   +     +   +                                          bFGF    0      +     0   +     0   0                                          IGF-I   +      +     +   +     +   +                                          IGF-II  +      +     +   +     +   +                                          TGF-B1  +      (-)   0   +     0   +                                          IL-1B   +      +     +   +     ND  +                                          LIF     +      0     +   0     ND  0                                          TNF-a   +      +     +   ND    ND  +                                          BMP-2,3,4,7                                                                           +      (-)   0   +     +   +                                          OSR-6                                                                         HepAg FT                                                                              0      0     0   0     0   ND                                         0.2M NaCl                                                                             +      +     0   0     0   ND                                         1.0M NaCl                                                                             0      +     0   0     +   ND                                         2.0M NaCl                                                                             +      0     0   0     0   ND                                         __________________________________________________________________________     Table Legend. Comparison of proliferative effects of a variety of mitogen     on target cells. 0 = no stimulation (equivalent to 0% serum control), + =     mitogenic stimulation at least 50% above the 0% serum control, - =            inhibition of proliferation at least 50% of 0% serum control, ND = not        determined.                                                              

The results of the mitogenicity assay (Table 2) indicate that OSR-6cells produce a number of growth factors that can be fractionated fromthe serum-free conditioned medium by heparin agarose chromatography.Based on the selective stimulation of the 0.2M NaCl, 1.0M NaCl, and 2.0MNaCl fractions, the identity of the growth factors present in thesefractions is not readily apparent, as none of the known growth factorstested show a similar profile of mitogenic activity for the target cellsin the panel. In particular, the mitogenic growth factor, or growthfactors, present in the 0-0.2M NaCl elute preferentially stimulated theproliferation of osteoblastic cells and not multipotential cells andfibroblasts.

EXAMPLE 1

OSR-6 cells were cultured in roller bottles (Corning, 850 cm² surfacearea) for the large scale generation of serum-free conditioned medium.Briefly, OSR-6 cells from a confluent 150 cm² flask were transferred toa roller bottle containing approximately 100 ml of growth medium and theroller bottle was sealed after introducing at atmosphere of 5% CO₂ /95%air. A total of 40 roller bottles were seeded with OSR-6 cells. Theroller bottle cell cultures were incubated at 37° C. using a rollerapparatus (Wheaton) adjusted to approximately 2 rpm of the rollerbottles. After the cells reached confluency, the growth medium wasdecanted from the roller bottle, the attached cells were rinsed twicewith approximately 50 ml of phosphate buffered saline, and 150 ml ofserum-free medium per roller bottle was added. The cell cultures weregased with the 5% CO₂ /95% air mixture and cultured with rotation at 37°for 48 hours. The conditioned serum-free medium was harvested, clarifiedby centrifugation, and stored frozen at -20° C. The cell cultures werereplenished with serum containing growth medium and cultured for twodays. This recovery period was followed by another cycle of incubationin serum-free medium, as described above. After the second recoveryperiod, the volume of serum-free medium was reduced to 100 ml per rollerbottle and the cell cultures were incubated for 24 hours, followed by atwo-day recovery period in serum containing growth medium. Fouradditional cycles of serum-free medium incubation followed by serumcontaining growth medium recovery were employed. The last three cyclesused 30 roller bottle cultures. A total of 29 liters of serum-freeconditioned media was collected.

HEPARIN AGAROSE ELUTION PROCEDURE

Ten liters of the frozen serum-free conditioned media harvested fromroller bottle cultures of OSR-6 cells were thawed and two liters weredirectly applied to a heparin agarose column (Type I, Sigma) with a bedvolume of approximately 500 ml. Because the column became clogged, theremaining 8 liters of conditioned media was mixed with the heparinagarose in two successive rounds of batch binding 4×1 liter ofconditioned media per 100 ml of heparin agarose. The conditioned mediaand heparin agarose were mixed by gently rocking the slurry for 2 hoursat 4° C. The heparin agarose was allowed to settle and the supernatantcarefully decanted. After the second round of batch binding, the heparinagarose was resuspended in approximately 100 ml of 25 mM tris buffer, pH8.0, per 100 ml of heparin agarose and combined with the remainingheparin agarose from the original column. A new column was poured andequilibrated with 25 mM Tris buffer, pH 8.0. Proteins binding to theheparin agarose were eluted with a 2 liter linear gradient of 25 mM Trisbuffer, pH 8.0, to 2.0 M NaCl, 25 mM tris buffer, pH 8.0. Approximately15 ml fractions were collected. Aliquots of 10 ml from the flow throughand 1.0 ml from three consecutive fractions of the gradient weredialyzed against H₂ O, and lyophilized. The lyophilized fractions wereresuspended in 2.0 ml of serum-free medium, sterilized by centrifugalfiltration (Centrex filters, Schleicher and Schuell), and assayed formitogenic activity. The osteoblastic OSR-2 cells (American Type CultureCollection No. CRL 11065) and the fibroblastic NIH-3T3 cells (AmericanType Culture Collection No. CRL 1658) were used as target cells. Themitogenic response of cells was quantitated using a [³ H]thymidineincorporated assay as previously described (Isfort (1990) Somatic CellMolec. Genet. 16:109-121). Briefly, target cells are plated at 2×10³cells/well in a 96 well microtiter plate and incubated overnight ingrowth medium to allow the cells to attach. The growth medium is removedand the cells are washed three times with phosphate buffered saline(GIBCO) followed by an 8 hour incubation in 50 ul/well of serum-freemedium. The heparin agarose fractions were added to the serum starvedcells in a volume of 50 ul of medium, with serum-free medium serving asa control and 20% FBS (10% FBS final concentration) serving as apositive mitogenic control. After 16 hours incubation, 10 ul ofserum-free medium containing 1 uci of [³ H]thymidine (Amersham, 5Ci/mmol, 185 MBq/mmol) were added to each well and the cells wereincubated for 6 additional hours. The cells were then harvested andlysed on glass fiber filters using a PHD cell harvester (CambridgeTechnology, Inc.) and the radioactivity in the samples was assayed byscintillation spectrophotometry. The data were calculated from the meandpms of quadruplicate samples of the experimental groups and expressedas the fold incorporation relative to the serum-free treated cells. Theheparin agarose fractionation of mitogenic growth factors from theserum-free conditioned medium of OSR-6 cells is shown in FIGS. 1a, 1band 1c. The fractions containing the various heparin binding growthfactors are indicated in FIG. 1b and FIG. 1c wherein "#1", "#2", "#3","#4", "#5" and "#6" refer to the first, second, third, fourth, fifth andsixth heparin binding growth factors, respectively. The results of thisanalysis, together with the previous results from the small scaleheparin agarose fraction of serum-free conditioned medium (Table 2) aresummarized in Table 3.

TABLE 3 Summary of Growth Factors Secreted by OSR-6 Cells

1. A non-heparin binding growth factor that stimulates the proliferationof fibroblasts and osteoblast cells.

2. A first heparin binding, osteoblast specific growth factor present inthe 0-0.2M NaCl eluate from heparin agarose chromatography whichpreferentially stimulates the proliferation of osteoblast cells.

3. A second heparin binding growth factor present in the 0.2-0.4M NaCleluate that stimulates the proliferation of fibroblasts and osteoblastcells.

4. A third heparin binding growth factor present in the 0.5-0.7M NaCleluate that stimulates the proliferation of osteoblast cells andfibroblasts.

5. A fourth heparin binding growth factor present in the 0.7-0.9M NaCleluate which stimulates the proliferation of osteoblast cells.

6. A fifth heparin binding growth factor present in the 1.0-1.2M NaCleluate which stimulates the proliferation of osteoblast cells andfibroblasts.

7. A sixth heparin binding growth factor present in the 1.5-1.8M NaCleluate which stimulates the proliferation of osteoblast cells andfibroblasts.

The invention has been described herein with reference to certainpreferred embodiments and examples. Obvious variations may appear tothose skilled in the art. Therefore, the invention is not to beconsidered limited thereto but only by the claims which follow.

What is claimed is:
 1. A cell line having all of the identifyingcharacteristics of American Type Culture Collection Accession No. CRL11069.